Field of the Invention
The present invention relates to the detection of a nucleotide variation on a target nucleic acid sequence by a PTOCE (PTO Cleavage and Extension) assay.
Description of the Related Art
DNA hybridization is a fundamental process in molecular biology and is affected by ionic strength, base composition, length of fragment to which the nucleic acid has been reduced, the degree of mismatching, and the presence of denaturing agents. DNA hybridization-based technologies would be a very useful tool in specific nucleic acid sequence determination and clearly be valuable in clinical diagnosis, genetic research, and forensic laboratory analysis.
However, the conventional methods and processes depending mostly on hybridization are very likely to produce false positive results due to non-specific hybridization between probes and non-target sequences. Therefore, there remain problems to be solved for improving their reliability.
Besides probe hybridization processes, several approaches using additional enzymatic reactions, for example, TaqMan™ probe method, have been suggested.
In TaqMan™ probe method, the labeled probe hybridized with a target nucleic acid sequence is cleaved by a 5′ nuclease activity of an upstream primer-dependent DNA polymerase, generating a signal indicating the presence of a target sequence (U.S. Pat. Nos. 5,210,015, 5,538,848 and 6,326,145). The TaqMan™ probe method suggests two approaches for signal generation: polymerization-dependent cleavage and polymerization-independent cleavage. In polymerization-dependent cleavage, extension of the upstream primer must occur before a nucleic acid polymerase encounters the 5′-end of the labeled probe. As the extension reaction continues, the polymerase progressively cleaves the 5′-end of the labeled probe. In polymerization-independent cleavage, the upstream primer and the labeled probe are hybridized with a target nucleic acid sequence in close proximity such that binding of the nucleic acid polymerase to the 3′-end of the upstream primer puts it in contact with the 5′-end of the labeled probe to release the label. In addition, the TaqMan™ probe method discloses that the labeled probe at its 5′-end having a 5′-tail region not-hybridizable with a target sequence is also cleaved to form a fragment comprising the 5′-tail region.
There have been reported some methods in which a probe having a 5′-tail region non-complementary to a target sequence is cleaved by 5′ nuclease to release a fragment comprising the 5′-tail region.
For instance, U.S. Pat. No. 5,691,142 discloses a cleavage structure to be digested by 5′ nuclease activity of DNA polymerase. The cleavage structure is exemplified in which an oligonucleotide comprising a 5′ portion non-complementary to and a 3′ portion complementary to a template is hybridized with the template and an upstream oligonucleotide is hybridized with the template in close proximity. The cleavage structure is cleaved by DNA polymerase having 5′ nuclease activity or modified DNA polymerase with reduced synthetic activity to release the 5′ portion non-complementary to the template. The released 5′ portion is then hybridized with an oligonucleotide having a hairpin structure to form a cleavage structure, thereby inducing progressive cleavage reactions to detect a target sequence.
U.S. Pat. No. 7,381,532 discloses a process in which the cleavage structure having the upstream oligonucleotide with blocked 3′-end is cleaved by DNA polymerase having 5′ nuclease activity or FEN nuclease to release non-complementary 5′ flap region and the released 5′ flap region is detected by size analysis or interactive dual label. U.S. Pat. No. 6,893,819 discloses that detectable released flaps are produced by a nucleic acid synthesis dependent, flap-mediated sequential amplification method. In this method, a released flap from a first cleavage structure cleaves, in a nucleic acid synthesis dependent manner, a second cleavage structure to release a flap from the second cleavage structure and the release flaps are detected. U.S. Pat. No. 7,309,573 disclose a method including formation of a released flap produced by a nucleic acid synthesis; extension of the released flap; cleavage of an oligonucleotide during extension of the flap and detection of a signal generated by the cleavage of the oligonucleotide.
By hybridization of fluorescence-labeled probes in a liquid phase, a plurality of target nucleic acid sequences may be simultaneously detected using even a single type of a fluorescent label by melting curve analysis. However, the conventional technologies for detection of target sequences by 5′ nuclease-mediated cleavage of interactive-dual labeled probes require different types of fluorescent labels for different target sequences in multiplex target detection, which limits the number of target sequences to be detected due to limitation of the number of types of fluorescent labels.
U.S. Pat. Appln. Pub. 2008-0241838 discloses a target detection method using cleavage of a probe having a 5′ portion non-complementary to a target nucleic acid sequence and hybridization of a capture probe. A label is positioned on the non-complementary 5′ portion. The labeled probe hybridized with the target sequence is cleaved to release a fragment, after which the fragment is then hybridized with the capture probe to detect the presence of the target sequence. In this method, it is necessary that an uncleaved/intact probe is not hybridized with the capture probe. For that, the capture probe having a shorter length has to be immobilized onto a solid substrate. However, such a limitation results in lower efficiency of hybridization on a solid substrate and also in difficulties in optimization of reaction conditions.
Therefore, there remain long-felt needs in the art to develop novel approaches for detection of a target sequence, preferably multiple target sequences, in a liquid phase and on a solid phase by not only hybridization but also enzymatic reactions such as 5′ nucleolytic reaction in a more convenient, reliable and reproducible manner. Furthermore, a novel target detection method not limited by the number of types of labels (particularly, fluorescent labels) is also needed in the art.
In the meantime, nucleotide variations are important in the research and clinical fields. Of them, single nucleotide polymorphisms (SNPs) are most commonly found in a human genome and serve as markers for disease-related loci and pharmacogenetics (Landegren et al., 1998; Roses, 2000). SNPs are found at the rate of approximately 1 per 1000 bp in a human genome and their total number is estimated about three millions. For the detection of nucleotide variations such as SNP, deletion, insertion and translocation, various allelic discrimination technologies have been reported.
The allele-specific TaqMan probe is designed such that it is hybridized only with perfectly matched target sequences in extension step of PCR. The TaqMan probe has a reporter molecule and a quencher molecule capable of quenching the fluorescent signal from the reporter molecule. The TaqMan probe hybridized with target sequences is digested by 5′ nuclease activity of Taq DNA polymerase and the reporter molecule and the quencher molecule are separated to generate a target signal. For allelic discrimination, 13-20 mer probes conjugated with minor groove binder (MGB) are used (Livak, et al., Genet. Anal. 14:143-149(1999)). Since the allelic discrimination method using the TaqMan probe employs not only hybridization reaction but also enzymatic reactions of 5′ nuclease activity, its specificity is enhanced. However, the method has serious troublesome such as difficulties in allelic-specific probe design and optimized reaction conditions which have to discriminate difference by one mismatch. In addition, the conjugate with MGB is one of troubleshootings in the allele-specific TaqMan probe.
Therefore, there remain long-felt needs in the art to develop novel approaches for detection of a nucleotide variation in a more convenient, reliable and reproducible manner, which is capable of being free from shortcomings of the conventional technologies.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.